1. Field of the Invention
This invention is concerned with the design of antagonists of the interleukin-4 (IL-4) specific receptor, mimetic peptides that are able to act as such antagonists and their use as pharmaceutical agents for the treatment of disorders that are at least partly induced or mediated by the action of interleukin-4, especially the binding of interleukin-4 to its receptor.
2. Description of Related Art
Interleukin-4 is a 15 KDa glycoprotein produced mainly by T helper lymphocytes type 2 (TH2) and to a lesser extent by basophils, mast cells and eosinophils. Interleukin4 binds to the IL-4 receptor at the surface of target cells forming a binary complex that then recruits the interleukin-2 (IL-2) receptor xcex3c chain, which is also part of several other cytokine receptor systems (1-4). Upon binding to this receptor system, IL-4 can elicit different responses depending on the type of target cell, and is therefore part of the so-called pleiotropic cytokine family (5, 6). IL-4 can activate genes involved in the proliferation of T-cells, thymocytes, fibroblasts and capillary endothelial cells (7-9). It has also been reported to regulate the morphology and cytoskeletal organisation of human vascular endothelial cells, and to induce the expression of 15-lipoxygenase in monocytes (10, 11). IL-4 can also promote macrophage development by stimulating the lineage restriction of bipotent Granulocyte-Macrophage colony forming cells (12). Another important action of IL-4 is the induction of CD8+cytotoxic T-cells (13). It has also been shown that tumors cells expressing IL4 are rejected in vivo by recruited host granulocytes and macrophages (14, 15). It influences as well B-cell growth and controls IgG class switching of B cells expressing IgM into two isotypes: IgG4 and IgE (16, 17). The message conveyed to the nucleus by IL-4 upon binding to the heterodimeric receptor can lead to the expression of cell surface proteins including the IgE low affinity receptor (CD23) and the MHCII (major histocompatibility complex II) (18, 19). Transgenic mice overexpressing IL-4 present symptoms typical of allergic disease states, providing direct evidence for the pathophysiological role of IL-4 (2O). It seems therefore evident that IL-4 itself, or IL-4 antagonists, may have a wide range of therapeutic applications, ranging from the treatment of allergic diseases to cancer therapy.
Allergic diseases afflict considerable parts of the population of developed countries and account for a good deal of expenses with public health services. Only a precise understanding of the cellular and molecular interactions at the basis of allergic responses will render these processes amenable to pharmaceutical control.
The allergic response developed by an individual will depend on the allergen and on the part of the body in which the allergen engages with the immune system. Allergic rhinitis is characterized by sneezing and strong congestion of the upper airways, while asthma arises in the aftermath of the obstruction and constriction of the bronchi. Perturbations of the gastrointestinal tract may also arise when the immune activity affects the contraction of smooth muscle: that surround the stomach and intestine walls (21).
When an allergen enters the body it is confronted with antigen presenting cells that are able to recognise its foreign nature, phagocytoze and degrade it. The resulting fragments are then presented to T-lymphocytes, mainly T helper lymphocytes type II (TH2). These cells secrete several cytokines, including IL-2, IL-6, IL-10, IL-13 and IL4. The response induced by IL-4 upon binding to its heterodimeric receptor complex involves an intrachromosomal rearrangement event that leads to immunoglobulin class switching of B plasma cells from IgM to IgG4 and IgE. IL-4 is also able to upregulate the expression of the IgE low affinity receptor (CD23) on mast cells and B cells and the expression of its own receptor on lymphocytes. The allergen-specific immunoglobulin E antibodies associate with their receptor on the surface of mast cells in tissue and on basophils circulating in blood. When an allergen binds to two IgE molecules it will bring together their receptors, which results in the activation of different signal transduction pathways involving several enzymatic systems. This culminates with the secretion of a plethora of molecules by mast cells including histamine, cytokines and lipid moleculesilike prostaglandins and leukotrines. These are indeed the agents responsible for the allergic symptoms. As more IL-4 is produced by mast cells, this perpetuates its presence at the site of inflammation, causing an explosive reaction which in some instances can lead to hypotensive shock and even death. Hypotensive shock is characterized by a drop in blood pressure accompanied by a dramatic reduction in the supply of oxygen to the heart and brain, that arise as a consequence of widespread vascular changes induced by histamine (21-23).
In view of what has been stated above it seems clear that IL-4 is a dominant cytokine in allergic inflammation, for it determines whether B-cells give rise to IgE or other types of antibodies. Consequently, drugs capable of interfering with the activity of IL-4 will help reduce IgE levels and therefore control allergic reactions.
The activity of IL-4 can be inhibited by preventing the interaction of IL-4 with its receptor system, thereby suppressing the intracellular signals that are at the basis of allergic disease. The strategies available up to date to block cytokine-receptor interactions involve the use of monclonal antibodies against a cytokine or its receptor, soluble receptors and cytokine receptor antagonists (24, 25). Receptor antagonists are proteins that are capable of binding cytokine receptors with high affinity but are incapable of inducing signal transduction and therefore do not generate a biological response. Often receptor antagonists can be generated by mutating the wild-type cytokine. In this way, cytokine-derived antagonists have been successfully obtained for IL-4, growth hormone, prolactin and IL-6 (26-29). For interleukin-1 a natural receptor antagonist has been reported and recently the crystal structure of this antagonist complexed with the interleukin-1 receptor has been determined (30, 31). Nevertheless, thus far no other natural cytokine receptor antagonists are known.
The therapeutic potential of soluble receptors and monoclonal antibodies has been shown to be rather limited. Soluble receptors bind cytokine ligands with lower affinity than their membrane bound counterparts and therefore, in order to achieve efficient inhibition of the cytokine response, intolerable levels of soluble receptors would have to be applied. Furthermore, antibody or soluble receptor-cytokine complexes tend to be cleared off the body at much slower rate than the ligand alone. These complexes will then accumulate in the circulation and constitute a depot form of the cytokine that can be released later on. Because of the high turnover rate of protein molecules in the human body repeated administration would be required, rendering the treatment expensive and often dangerous because of possible immunogenicity of these proteins (24, 25, 32). For these reasons, much hope has been given to the advent of the above mentioned cytokine-derived antagonists as efficient therapeutical molecules. This new generation of biopharmaceuticals was expected to be of lower toxicity compared to other substances (33). However, recombinant cytokines are difficult to produce in large amounts in a cost-effective way. Most of the recombinant therapeutic proteins and vaccines available on the market are produced by large-scale fermentation of Escherichia coli carrying a gene coding for the protein of interest. In the particular case of cytokines and growth factors, including IL-4, this production strategy is hindered by the fact that these proteins generally form inclusion bodies when overexpressed in E. coli. The purification of the cytokine, or cytokine derivative, from inclusion bodies requires the use of chaotropic agents like guanidinium chloride, urea or strong detergents. An in vitro renaturation step then follows during which the protein is expected to fold into its three-dimensional structure (34). Although many proteins have been reported to have been successfully refolded from inclusion bodies, in the case of cytokines the yield of correctly folded protein is usually very low (35). Therefore, in vitro renaturation processes remain inefficient and expensive and it is quite difficult to achieve a perfect separation of the properly folded protein from: certain misfolded forms. This fact poses a serious drawback to the use of these proteins for therapeutic purposes, since even small amounts of improperly folded protein can be immunogenic. But this is not the only disadvantage that places cytokine derived therapeutics in a rank far from ideal medicines. All the inconveniences associated with protein drugs, like short serum half-life due to rapid proteolysis by serum proteolytic enzymes, low oral availability, and low local effectiveness as a consequence of systemic administration, have to be added to the list (33, 36). In principle, gene therapy is expected to circumvent most of these problems by making possible the expression proteins within the body at specific sites under tight control (37). However, gene therapy technology is still in its infancy, and meanwhile alternative therapeutic solutions have to be put forward.
In recent years, advancements in structural biophysical techniques, like Nuclear Magnetic Resonance (NMR), X-ray crystallography or Electron Microscopy, have made possible the determination of protein or protein complexes three-dimensional structures at high resolution. Additionally, NMR also offers the possiblity of investigating the dynamic properties of biomacromolecules in solution (38, 39). In the last years the three-dimensional structure of several cytokines and growth factors, like IL-2, IL-4, IL-6, GM-CSF, and hGH, has been determined by X-ray crystallography or NMR (40-51). The structure of these cytokines consists of four helices connected by two long loops containing short segments of xcex1-helix or xcex2-sheet structure. Such an arrangement of four helices forms the so-called four-helix bundle structural motif. All the cytokines listed above display an up-up-down-down-topoloy of the helices (52).The structure of the complex of the hGH bound to its homodimeric receptor and to the prolactin receptor has also been made available (53-55). Simultaneously, a great effort has been devoted to the mapping of the putative receptor binding epitopes by site-directed mutagenesis (56, 57). The comparison of the mutagenesis and structural data is of pivotal importance to the understanding of the structure-function relationship of cytokine-receptor systems and allows the identification of crucial intermolecular interactions at the active site. The structure of the human growth hormone-receptor complex shows that despite the fact that a large surface area is buried both on the hormone and on the receptor upon binding, corresponding to approximately 33 side chains on both molecules, only 9 residues contribute significantly to the binding energy (58-60). These data suggest that functional epitopes mediating growth factor or cytokine-receptor interaction may be rather small, and therefore there is a good chance that they can be emulated by rationally designed small peptide molecules. These peptidomimetics should be able to play the role of larger polypeptide ligands in recognising and/or activating receptor targets, and would be used as or developed into potent cytokine or cytokine receptor agonist or antagonist drugs.
When it comes to the development of therapeutic peptides researchers are confronted with the challenge of finding the best possible candidate that is able to mediate the desired bilogical effect in the most efficient way. This is usually achieved by creating libraries of compounds that have diverse molecular shapes and functional characteristics. Phage display technology has been of precious help in the creation and screening of vast peptide libraries (61-63). This methodology has been successfully used to isolate peptide mimetics of erythropoietin (EPO). One of these peptides is able to bind the EPO receptor with a 0.2 xcexcM affinity constant (Kd) and the three-dimensional structure of this peptide in complex with the EPO receptor has also been determined (64, 65). The small peptide dimerizes forming a four-stranded anti-parallel xcex2-sheet that is able to bind two EPO receptor molecules and induce a response similar to erythropoietin. Phage display has also been used to imp rove the stability and affinity of a two-helix derivative of the three-helix Z-domain of protein A. This 59 residue three-helix bundle binds the Fc portion of Immunoglobulin G (IgG) with a Kd of 10 nM. By using a combination of phage display and structural data the binding domain has been reduced to a 33 residue peptide that is able to bind IgG with virtually the same affinity as the wild-type protein (66). The 15-residue atrial natriuretic peptide (ANP) mimetic is another example of a synthetic peptide that has been selected by phage display to specifically bind a receptor molecule (67).
The present invention relates to the rational design of an interleukin-4 mimetic peptide using methodologies herein described below.